+

US7063782B2 - Electrochemical detection of ischemia - Google Patents

Electrochemical detection of ischemia Download PDF

Info

Publication number
US7063782B2
US7063782B2 US10/304,610 US30461002A US7063782B2 US 7063782 B2 US7063782 B2 US 7063782B2 US 30461002 A US30461002 A US 30461002A US 7063782 B2 US7063782 B2 US 7063782B2
Authority
US
United States
Prior art keywords
ischemic event
sample
ion
albumin
diagnosed
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime, expires
Application number
US10/304,610
Other languages
English (en)
Other versions
US20030132125A1 (en
Inventor
Hollie Wayment
Gary Fagan
Peter A. Crosby
Shannon George
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ischemia Technologies Inc
Original Assignee
Ischemia Technologies Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ischemia Technologies Inc filed Critical Ischemia Technologies Inc
Priority to US10/304,610 priority Critical patent/US7063782B2/en
Assigned to ISCHEMIA TECHNOLOGIES, INC. reassignment ISCHEMIA TECHNOLOGIES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CROSBY, PETER A., FAGAN, GARY, GEORGE, SHANNON, WAYMENT, HOLLIE
Assigned to SILICON VALLEY BANK reassignment SILICON VALLEY BANK SECUIRITY AGREEMENT Assignors: ISCHEMIA TECHNOLOGIES, INC.
Publication of US20030132125A1 publication Critical patent/US20030132125A1/en
Assigned to ISCHEMIA TECHNOLOGIES, INC. reassignment ISCHEMIA TECHNOLOGIES, INC. RELEASE OF SECURITY INTEREST Assignors: SILICON VALLEY BANK
Assigned to GENERAL ELECTRIC CAPITAL CORPORATION, AS AGENT reassignment GENERAL ELECTRIC CAPITAL CORPORATION, AS AGENT SECURITY INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ISCHEMIA TECHNOLOGIES, INC.
Priority to US11/415,929 priority patent/US20060191788A1/en
Application granted granted Critical
Publication of US7063782B2 publication Critical patent/US7063782B2/en
Assigned to GENERAL ELECTRIC CAPITAL CORPORATION, AS AGENT reassignment GENERAL ELECTRIC CAPITAL CORPORATION, AS AGENT FIRST LIEN INTELLECTUAL PROPERTY SECURITY AGREEMENT Assignors: ADVANTAGE DIAGNOSTICS CORPORATION, A DELAWARE CORPORATION, APPLIED BIOTECH, INC., A CALIFORNIA CORPORATION, BINAX, INC., A DELAWARE CORPORATION, FIRST CHECK DIAGNOSTICS CORP., A DELAWARE CORPORATION, FOREFRONT DIAGNOSTICS, INC., A CALIFORNIA CORPORATION, IM US HOLDINGS, LLC, A DELAWARE LIMITED LIABILITY COMPANY, INCA ACQUISITION, INC., A DELAWARE CORPORATION, INNOVACON, INC., A DELAWARE CORPORATION, INNOVATIONS RESEARCH, LLC, A DELAWARE LIMITED LIABILITY COMPANY, INNOVATIVE MOBILITY, LLC, A FLORIDA LIMITED LIABILITY COMPANY, INSTANT TECHNOLOGIES, INC., A VIRGINIA CORPORATION, INVERNESS MEDICAL - BIOSTAR, INC., A DELAWARE CORPORATION, INVERNESS MEDICAL INNOVATIONS, INC., A DELAWARE CORPORATION, INVERNESS MEDICAL INTERNATIONAL HOLDING CORP. II, A DELAWARE CORPORATION, INVERNESS MEDICAL INTERNATIONAL HOLDING CORP., A DELAWARE CORPORATION, INVERNESS MEDICAL, LLC, A DELAWARE LIMITED LIABILITY COMPANY, ISCHEMIA TECHNOLOGIES, INC., A DELAWARE CORPORATION, IVC INDUSTRIES, INC., A DELAWARE CORPORATION, OSTEX INTERNATIONAL, INC., A WASHINGTON CORPORATION, QUALITY ASSURED SERVICES, INC., A FLORIDA CORPORATION, SELFCARE TECHNOLOGY, INC., A DELAWARE CORPORATION, SPDH, INC., A DELAWARE CORPORATION, UNIPATH ONLINE, INC., A MASSACHUSETTS CORPORATION
Assigned to GENERAL ELECTRIC CAPITAL CORPORATION, AS AGENT reassignment GENERAL ELECTRIC CAPITAL CORPORATION, AS AGENT SECOND LIEN INTELLECTUAL PROPERTY SECURITY AGREEMENT Assignors: ADVANTAGE DIAGNOSTICS CORPORATION, A DELAWARE CORPORATION, APPLIED BIOTECH, INC., A CALIFORNIA CORPORATION, BINAX, INC., A DELAWARE CORPORATION, FIRST CHECK DIAGNOSTICS CORP., A DELAWARE CORPORATION, FOREFRONT DIAGNOSTICS, INC., A CALIFORNIA CORPORATION, IM US HOLDINGS, LLC, A DELAWARE LIMITED LIABILITY COMPANY, INCA ACQUISITION, INC., A DELAWARE CORPORATION, INNOVACON, INC., A DELAWARE CORPORATION, INNOVATIONS RESEARCH, LLC, A DELAWARE LIMITED LIABILITY COMPANY, INNOVATIVE MOBILITY, LLC, A FLORIDA LIMITED LIABILITY COMPANY, INSTANT TECHNOLOGIES, INC., A VIRGINIA CORPORATION, INVERNESS MEDICAL - BIOSTAR, INC., A DELAWARE CORPORATION, INVERNESS MEDICAL INNOVATIONS, INC., A DELAWARE CORPORATION, INVERNESS MEDICAL INTERNATIONAL HOLDING CORP. II, A DELAWARE CORPORATION, INVERNESS MEDICAL INTERNATIONAL HOLDING CORP., A DELAWARE CORPORATION, INVERNESS MEDICAL, LLC, A DELAWARE LIMITED LIABILITY COMPANY, ISCHEMIA TECHNOLOGIES, INC., A DELAWARE CORPORATION, IVC INDUSTRIES, INC., A DELAWARE CORPORATION, OSTEX INTERNATIONAL, INC., A WASHINGTON CORPORATION, QUALITY ASSURED SERVICES, INC., A FLORIDA CORPORATION, SELFCARE TECHNOLOGY, INC., A DELAWARE CORPORATION, SPDH, INC., A DELAWARE CORPORATION, UNIPATH ONLINE, INC., A MASSACHUSETTS CORPORATION, WAMPOLE LABORATORIES, LLC, A DELAWARE LIMITED LIABILITY COMPANY
Assigned to GENERAL ELECTRIC CAPITAL CORPORATION reassignment GENERAL ELECTRIC CAPITAL CORPORATION SECURITY AGREEMENT Assignors: ADVANTAGE DIAGNOSTICS CORPORATION, ALERE MEDICAL INCORPORATED, ALERE SAN DIEGO, INC., ALERE SCARBOROUGH, INC., AMEDITECH INC., APPLIED BIOTECH, INC., BINAX, INC., BIOSITE INCORPORATED, CHOLESTECH CORPORATION, GENECARE MEDICAL GENETICS CENTER, INC., HEMOSENSE, INC., INSTANT TECHNOLOGIES, INC., INVERNESS MEDICAL - BIOSTAR INC., ISCHEMIA TECHNOLOGIES, INC., MARTIA HEALTHCARE, INC., MATRITECH, INC., ZYCARE INC.
Assigned to APPLIED BIOTECH, INC., ALERE SAN DIEGO, INC., HEMOSENSE, INC., ADVANTAGE DIAGNOSTICS CORPORATION, AMEDITECH INC., MATRIA HEALTHCARE, INC., ALERE SCARBOROUGH, INC., BINAX, INC., MATRITECH, INC., ALERE MEDICAL, INC., INSTANT TECHNOLOGIES, INC., CHOLESTECH CORPORATION, BIOSITE INCORPORATED, INVERNESS MEDICAL - BIOSTAR INC., ZYCARE, INC., ISCHEMIA TECHNOLOGIES, INC., GENECARE MEDICAL GENETICS CENTER, INC. reassignment APPLIED BIOTECH, INC. NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287 Assignors: GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT
Assigned to GENERAL ELECTRIC CAPITAL CORPORATION, AS COLLATERAL AGENT reassignment GENERAL ELECTRIC CAPITAL CORPORATION, AS COLLATERAL AGENT INTELLECTUAL PROPERTY SECURITY AGREEMENT Assignors: ALERE CONNECT, LLC, ALERE SAN DIEGO, INC. (FKA BIOSITE INC. OR FKA CHOLESTECH CORP. OR FKA HEMOSENSE INC. OR FKA INVERNESS MEDICAL-BIOSTAR INC. OR FKA ISCHEMIA TECHNOLOGIES, INC. OR FKA TWISTDX, INC.), ALERE SCARBOROUGH, INC. (FKA MATRITECH, INC. FKA ADVANTAGE DIAGNOSTICS CORP. OR FKA BINAX, INC. OR FKA MILANO ACQUISITION CORP.), ESCREEN, INC., INNOVACON, INC. (FKA APPLIED BIOTECH, INC. OR FKA AMEDITECH INC.), IONIAN TECHNOLOGIES, LLC (FKA IONIAN TECHNOLOGIES, INC.), QUALITY ASSURED SERVICES INC. (FKA ZYCARE INC.), STANDING STONE, LLC
Assigned to HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS SUCCESSOR ADMINISTRATIVE AGENT reassignment HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS SUCCESSOR ADMINISTRATIVE AGENT ASSIGNMENT OF IP SECURITY AGREEMENT, PREVIOUSLY RECORDED AT REEL 036994, FRAME 0192 Assignors: GENERAL ELECTRIC CAPITAL CORPORATION, AS RETIRING ADMINISTRATIVE AGENT
Assigned to ALERE SAN DIEGO, INC. (FKA BIOSITE INC. OR FKA CHOLESTECH CORP. OR FKA HEMOSENSE INC. OR FKA INVERNESS MEDICAL-BIOSTAR INC. OR FKA ISCHEMIA TECHNOLOGIES, INC. OR FKA TWISTDX, INC.), INNOVACON, INC. (FKA APPLIED BIOTECH, INC. OR FKA AMEDITECH INC.), IONIAN TECHNOLOGIES, LLC (FKA IONIAN TECHNOLOGIES, INC.), ALERE SCARBOROUGH, INC. (FKA MATRITECH, INC. FKA ADVANTAGE DIAGNOSTICS CORP. OR FKA BINAX, INC. OR FKA MILANO ACQUISITION CORP.), QUALITY ASSURED SERVICES INC. (FKA ZYCARE INC.), ESCREEN, INC., ALERE CONNECT, LLC, STANDING STONE, LLC reassignment ALERE SAN DIEGO, INC. (FKA BIOSITE INC. OR FKA CHOLESTECH CORP. OR FKA HEMOSENSE INC. OR FKA INVERNESS MEDICAL-BIOSTAR INC. OR FKA ISCHEMIA TECHNOLOGIES, INC. OR FKA TWISTDX, INC.) RELEASE OF SECURITY INTEREST IN INTELLECTUAL PROPERTY RECORDED AT REEL 036994, FRAME 0192 AND REEL 037115, FRAME 0498 Assignors: HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS COLLATERAL AGENT
Adjusted expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
    • G01N2333/765—Serum albumin, e.g. HSA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/49—Systems involving the determination of the current at a single specific value, or small range of values, of applied voltage for producing selective measurement of one or more particular ionic species

Definitions

  • This invention pertains to in vitro diagnostic devices using electrochemical detection of an analyte.
  • this invention uses redox reactions, as revealed by amperometric and potentiometric measurements in all forms (coulometry, voltammetry, ion selective electrode, chronoamperometry etc.), as an indication of an ischemic event by detecting all modifications to albumin.
  • Ischemia is the state of imbalance of oxygen supply and demand in a part of the body often due to a constriction or an obstruction in the blood vessel supplying that part.
  • the two most common forms of ischemia are cardiovascular and cerebrovascular.
  • Cardiovascular ischemia is generally a direct consequence of coronary artery disease, and is usually caused by rupture of an atherosclerotic plaque in a coronary artery, leading to formation of thrombus (blood clot), which can occlude or obstruct a coronary artery, thereby depriving the downstream heart muscle of oxygen. Prolonged ischemia can lead to cell death or necrosis, and the region of dead tissue is commonly called an infarct. Patients suffering an event of acute cardiac ischemia often present to a hospital emergency room with chest pain and other symptoms and signs, such as changes to an electrocardiogram (ECG). This type of presentation is referred to as Acute Coronary Syndrome (ACS). A patient diagnosed with ACS requires immediate treatment to avoid irreversible damage to the heart muscle.
  • ECG electrocardiogram
  • Cerebral ischemia is often due to narrowing of the arteries leading to the brain.
  • TIA Transient Ischemic Attack
  • CVA cerebrovascular accident
  • the continuum of ischemic disease includes five conditions: (1) elevated blood levels of cholesterol and other lipids; (2) build-up of atherosclerotic plaque and subsequent narrowing of the arteries; (3) reduced blood flow to a body organ (as a result of arterial narrowing or plaque rupture and subsequent thrombus formation); (4) cellular damage to an organ caused by a lack of oxygen; (5) death of organ tissue caused by sustained oxygen deprivation. Stages three through five are collectively referred to as “ischemic disease,” while stages one and two are considered its precursors. It is important to distinguish between the state of ischemia and the disease that leads to it. For example, a patient with coronary artery disease is not always in the state of cardiac ischemia, but a person in the state of cardiac ischemia almost invariably suffers from coronary artery disease.
  • ischemic heart disease accounted for 778,000 deaths in the U.S. in 1998 (2002 Heart and Stroke Statistical Update, 2002 American Heart Association). Additionally, as many as 3 to 4 million Americans suffer from what is referred to as “silent ischemia.” This is a condition where ischemic heart disease is present without the usual and classic symptoms of chest pain or angina.
  • Cardiac enzymes specifically the creatine kinase isoenzyme (CK-MB) and other markers of cardiac necrosis, specifically myoglobin and the Troponin I and Troponin T biochemical markers, are utilized for diagnosing heart muscle injury.
  • CK-MB creatine kinase isoenzyme
  • these enzymes and markers are only capable of detecting the existence of cell death or necrosis, and therefore have limited or no value in patients who have ischemia without necrosis, such as those in an ischemic state prior to myocardial infarction.
  • these enzymes and markers do not show a measurable increase until several hours after the onset of necrosis.
  • the cardiac troponins do not show a measurable increase above normal in a person's blood test until about four to six hours after the beginning of a heart attack and do not reach peak blood level until about 18 hours after such an event.
  • the primary shortcoming of using markers of cardiac necrosis for diagnosis of ischemic states is that these markers are only detectable after heart tissue has been irreversibly damaged.
  • Diagnosis of coronary artery disease is done either by imaging (e.g., coronary angiography) or by provocative testing, where the intent is to deliberately induce cardiac ischemia and observe the effects.
  • imaging e.g., coronary angiography
  • provocative testing where the intent is to deliberately induce cardiac ischemia and observe the effects.
  • the patient is exercised at an increasing rate to see if symptoms of ischemia are evoked, or if changes indicative of ischemia can be observed on the ECG.
  • Stress ECG is commonly used as an initial screen for coronary artery disease, but is limited by its accuracy rates of only 25–50% (see for example, Froemaschine, V F et al. (1988) Ann. Intern. Med. 128(12):965–974).
  • myocardial perfusion imaging in which a radioactively tagged chemical is injected during stress testing. Normally metabolizing cardiac tissue is able to take up the radioactively tagged chemical, and is visualized using conventional imaging techniques (PET or SPECT scanning) thereby allowing differentiation between viable and damaged cardiac tissue.
  • the present invention is believed to be advantageous over the known methods of diagnosis in that it is a simple blood test which will offer comparable accuracy at far lower costs and decreased risk and inconvenience to the patient. It is believed that the present invention provides specificity and sensitivity levels that are comparable in accuracy to current diagnostic standards.
  • ischemia Although there are well established biochemical markers of myocardial necrosis which can be detected in a blood sample using a point of care (POC) instrument, other than as described below in relation to the ACBTM Test, there are no well established biochemical markers for ischemia, and presently no POC instrument for detection of ischemia.
  • An ideal test would be a blood test, preferably administered with a small, simple device providing quick, accurate results that can be used to test for disease, for example at the bedside of a patient with minimal amount of discomfort.
  • HSA human serum albumin
  • Exposure of HSA to ischemic tissue produces modifications to the N-terminus (Bar-Or, D. et al. (2000) J. Emerg. Med. 19:311–315; PCT/US99/22905), and possibly other sites, on the albumin molecule.
  • the N-terminus of albumin has been well characterized as being the primary binding site for several transition metals such as cobalt, nickel and copper (Sadler, P. et al. (1994) Eur. J. Biochem. 220:193–200; Lakusta, H. et al. (1979) J. Inorg. Biochem. 11:303–315; Gasmi, G. et al. (1997) J.
  • a known amount of a transition metal is added to a biological sample (patient sample comprised of whole blood, serum or plasma, urine, cerebrospinal fluid, saliva and the like), normal albumin and IMA can be differentiated by monitoring the amount of non-binding metal.
  • Metal added to the sample will be sequestered at the N-terminus and possibly other sites on albumin more frequently in a non-ischemic sample than in an ischemic sample in which albumin has been modified in such a way that it can no longer bind the metal.
  • the metal not sequestered at the N-terminus and possibly other sites on albumin in the samples can then be detected and quantified using the Albumin Cobalt Binding (ACB) Test (Ischemia Technologies, Inc., Denver, Colo.), which, as described in PCT/US99/22905, filed Oct. 1, 1999, and U.S. Pat. No. 5,227,307, uses calorimetric methods to determine the amount of IMA present in the sample.
  • PCT/US99/22905 also provides a detailed description of the N-terminal modifications to albumin during an ischemic event. Studies have been conducted demonstrating the clinical utility of IMA via the ACB test in diagnosing and risk stratifying patients.
  • the ACB Test uses a laboratory chemistry analyzer to quantify IMA, but presently, there are no POC tests for ischemia.
  • An ideal test would be a blood test, preferably administered with a small, simple device providing quick, accurate results referenced to a standard curve with a quality control system that can be used to test for disease at the bedside of a patient with minimal amount of discomfort.
  • the aim of this invention is to provide such a diagnostic test.
  • Another object is to provide a diagnostic test that determines a difference in current or potential measurements in biological fluids from ischemic patients and non-ischemic individuals, wherein the samples are first combined with cobalt or another transition metal.
  • Another object of the subject invention is to use data processing techniques to identify features of the electrochemical output data from an electrochemical assay for determining the differences between ischemic and non-ischemic individuals.
  • the subject invention concerns the electrochemical detection of non-sequestered transition metal ion in a biological sample to which a known amount of transition metal has been added.
  • the amount of non-sequestered metal ion reflects the degree of ischemia modified albumin, which in turn can be used to determine the occurrence or nonoccurrence of an ischemic event.
  • the subject invention concerns a method for in vitro detection or measurement of albumin derivatives, which can be diagnostic of an ischemic event.
  • This method comprises the steps of: providing a patient sample comprising albumin and/or derivatives thereof in a vessel that is connected to an electrochemistry apparatus; operating the electrochemistry apparatus to utilize an electrochemical technique; optionally offsetting a background characteristic electrochemical output signal of the sample; adding a known (optionally excess) amount of a transition metal to the sample, whereby at least some of the ion binds to said albumin metal sequestering sites and remaining ion is non-sequestered; optionally adding an indicator or amplifier to the sample to bind to the non-sequestered ion; measuring the characteristic electrochemical output signal associated with the non-sequestered ion; optionally using a standard curve to convert the output signal into a value; whereby the measured or the converted value, if it exceeds a predetermined value, can be diagnostic of an ischemic event.
  • the characteristic electrochemical output signal associated with the
  • the subject invention comprises a method for in vitro detection or measurement of albumin derivatives, which can be diagnostic for an ischemic event comprising: providing a patient sample comprising albumin and/or derivatives or modifications thereof in a vessel having a reference electrode, a working electrode and an optional auxiliary electrode, said electrodes being connected to a potentiostat; operating the potentiostat to apply a potential to the sample; optionally offsetting a background current from redox reactions in the sample to zero; adding a known (optionally excess) amount of a transition metal ion to the sample, whereby at least some of the ion binds to said albumin N-terminus and possibly other sequestering sites and the remaining ion is non-sequestered; optionally providing an indicator or amplifier to the sample to bind to the non-sequestered metal ion; permitting the reaction of ion and albumin to reach equilibrium or a predetermined processing time; and measuring the current associated with the non-sequestered ion.
  • the measured current can then be referenced to a standard curve and converted to a value.
  • the measured current, or the converted value is related to the concentration of the non-sequestered metal ion, and is therefore related to the amount of IMA.
  • a predetermined value can be established, below which the sample can be said to be non-ischemic, and above which the sample can be said to be ischemic.
  • non-sequestered metal ion In measuring the non-sequestered metal ion, it is possible to measure not only free metal, i.e., metal not bound to albumin, but also metal ion that is bound to albumin, but in such a manner as to remain non-sequestered from detection.
  • non-sequestered ions refer not only to free ion that is not bound to albumin, but also to ions that are loosely associated with albumin but which remain non-sequestered.
  • Non-sequestered metal ions may be measured directly by electrochemical techniques or using an indicator or amplifier complexes such as dimethylglyoxime (DMG), dithiothreitol (DTT) or common covalent chelating agents such as EDTA and the like, according to methods known in the art.
  • Indicators or amplifiers may be incorporated into the electrode amalgam, or may be added to the sample vessel before, during or after addition of the metal ion. Indicators and amplifiers may also be attached to a solid-phase in such a manner as to remain capable of binding metal ions, thereby allowing for phase separation of the indicator/amplifier signal from the reaction mixture.
  • the indicators/amplifiers bind to the non-sequestered metal ion and permit the measurement of the metal ion in the current embodiment of the subject invention. While the detection of current associated with non-sequestered metal ions may be enhanced by the use of indicators or amplifiers, it may also be possible to detect the current associated with non-sequestered metal ions without indicators or amplifiers.
  • the subject invention comprises a method for in vitro detection or measurement of albumin derivatives which may be diagnostic for an ischemic event comprising: providing a patient sample comprising albumin and/or derivatives or modifications thereof in a vessel having an ion selective electrode and a reference electrode, wherein the two electrodes are connected to an electrostat or a voltmeter, and wherein the ion selective electrode comprises a barrier that is selectively permeable to a transition metal ion; operating the electrostat/voltmeter to measure the potential difference between the reference electrode and the ion selective electrode in contact with the sample; optionally offsetting the background potential difference of the sample; adding a known (optionally excess) amount of the transition metal ion to the sample, whereby at least some of the ion binds to the N-terminus and possibly other sequestering sites of the albumin and the remaining ion is non-sequestered; optionally providing an indicator or amplifier to the sample to bind with the non-sequestered metal ion; permitting the non-
  • This voltage change can be related to a standard calibration curve and converted to a value.
  • the measured voltage, or the converted value is related to the concentration of the non-sequestered metal ion, and is therefore related to the amount of IMA.
  • a predetermined value can be established, below which the sample can be said to be non-ischemic, and above which the sample can be said to be ischemic.
  • the subject invention also comprises a device for in vitro electrochemical detection or measurement of albumin derivatives which can be diagnostic for an ischemic event comprising: an electronics module housing having a display means, a data entry and control means, an aperture, internal contacts in said aperture for electrical continuity with the display means, a power source, and optionally a link to a laboratory information system; and a sample analysis strip comprising a sample well, and a plurality of electrodes, wherein each electrode is positioned in the sample well and connected via a corresponding trace to a corresponding contact on the strip; wherein insertion of the strip into the module housing engages each strip contact with its corresponding module contact, which permits electrical continuity with the display means, whereby the albumin derivatives may be detected and/or measured.
  • the link to a laboratory information system is possible via several means, including a direct electrical connection, an infrared link or a wireless link. This device can be used with either the current or the potential difference embodiments for detection of an ischemic event in a biological sample.
  • the invention provides a device for in vitro electrochemical detection or measurement of albumin derivatives in a sample which may be diagnostic for an ischemic event.
  • This device comprises: a housing having an aperture for receiving a sample analysis strip, the sample analysis strip having a sample well, a plurality of electrodes positioned in the sample well and a like plurality of first contacts interconnected to the electrodes; control means within the housing; and a plurality of second contacts in the aperture electrically coupled with the control means for interconnecting with the plurality of first contacts when the sample analysis strip is inserted within the aperture.
  • each first contact engages a corresponding second contact whereby the albumin derivatives can be detected or measured by said control means.
  • This device can include a link to a laboratory information system., which may be a direct electrical connection, an infrared link or a wireless link.
  • the device may also have display means within the housing which is electrically coupled with the control means; the display means displays information indicative of detected albumin derivatives.
  • the device may also have data entry means electrically coupled to the control means.
  • the control means includes means for measuring a characteristic electrochemical output signal associated with the sample in the sample analysis strip; and means for determining whether the electrochemical output signal exceeds a predetermined value, whereby an ischemic event is indicated.
  • the electrochemical output signal can be a current associated with non-sequestered metal ion in the sample to which has been added a known amount of a transition metal ion.
  • the electrochemical output signal can be a potential difference measured by an ion selective electrode which has a barrier that is selectively permeable to a transition metal ion, where the potential difference is measured in sample to which a known amount of a transition metal ion has been added.
  • FIG. 1 illustrates the components required for an electrochemical measuring device.
  • FIG. 2 illustrates the basic components of a functioning potentiostat.
  • FIG. 3 shows a graph of cobalt dependent current peaks measured by an adsorption technique.
  • FIG. 4 shows a graph of the cobalt dependent current peaks measured by a complexing ligand using a voltammetry technique.
  • FIG. 5 shows a data output profile of a cobalt current response measured by amperometry.
  • FIG. 6 shows a cobalt dependent current response measured by cyclic voltammetry.
  • FIG. 7 shows a cobalt dependent response in the presence of increasing concentrations of albumin.
  • FIG. 8 shows a cobalt dependent response in the presence of increasing concentrations of copper.
  • FIG. 9 shows a significant population difference between normal and modified albumin present in a biological fluid as measured by cyclic voltammetry.
  • FIG. 10 illustrates a hand held, point of care device suitable for practicing the methods of the subject invention.
  • IMA refers to ischemia induced modifications to albumin that affect the transition metal binding capacity of the molecule.
  • IMA includes albumin N-terminal derivatives, albumin with derivatized non N-terminal metal binding sites, which when non-derivatized, are capable of sequestering metals from detection, and full length albumin that has a copper occupied N-terminus.
  • albumin N-terminal derivatives refers to those species of albumin that are altered or truncated at the N-terminus as a result of an ischemic event. Specifically, the derivatives include those albumin species lacking 4, 3, 2 and 1 N-terminal amino acids, as well as a full-length albumin that is acetylated or otherwise derivatized at its terminal Asp residue. Albumin-terminal derivatives cannot form albumin-metal complexes and may be found in the blood of ischemic patients.
  • Excess amount of metal ion or “excess metal ion” refers to addition of an amount of metal ion that will substantially exceed the stoichiometrically available albumin metal ion binding sites such that substantially all full-length albumin is bound to metal ion at its N-terminus and substantially all albumin is bound to metal ion at other metal binding/sequestering sites, if any.
  • an “indicator”, “amplifier” or “complexing agent” is a compound that forms a complex with the transition metal that is non-specifically bound to the albumin, e.g., at non-N-terminus functional sites including thiol groups.
  • indicators or amplifiers are DMG, DTT and common divalent chelating agents such as ethylenediaminetetraacetic acid (EDTA) and the like. These compounds permit or enhance the measurement of the contribution of the non-sequestered metal ions that are loosely associated with albumin, to the current or voltage in the subject embodiments. See, e.g., Lee et al. (1980) Am. J. Med. Technol. 46(12):853–7; and Wang et al. (2000) Anal. Chem. 72(14):3218–22.
  • Non-sequestered ions as discussed above, means free ions that are not bound to albumin, and ions that are loosely associated with albumin but which remain non-sequestered, i.e., capable of measurement by direct or indirect electrochemical techniques. Indirect electrochemical techniques involve the use of complexing agents.
  • Patient sample means any patient sample that contains albumin (full length and/or derivatives), and can include whole blood, serum, plasma, other blood fraction, urine, saliva, cerebrospinal fluid, breast milk or the like.
  • Predetermined processing time refers to a selected time after addition of the transition metal at which measurements are consistently taken. The predetermined processing time may be shorter than the time required for the achievement of equilibrium.
  • Predetermined value refers to that current value, potential difference value, a value derived from a standard curve or the like that has been determined by clinical tests with normal individuals and ischemic patients to be the cutoff value above which an ischemic event is indicated.
  • “Sequestered” ions are metal ions that are coordinated (bound) to albumin in such a manner as to be rendered incapable of reacting with a complexing agent or in such a manner as to prevent the complexing agent from stripping the bound metal from albumin.
  • Transition metal refers to any transition metal of the Periodic Table.
  • a preferred transition metal is cobalt.
  • albumin modified at the N-terminus and possibly other metal sequestering sites can be differentiated from non-modified albumin by monitoring the current resulting from redox reactions of transition metals, or potentiometrically as with an ion selective electrode. Both systems require only that the metal analyte bound at the N-terminus and possibly other metal sequestering sites of albumin not be detectable electrochemically.
  • a current measurement resulting from a redox reaction can be made by applying a potential specific to the analyte of interest to the system.
  • a known concentration of a metal is introduced to the sample.
  • the time interval for current stabilization will be dependent on the binding kinetics of the metal to the metal sequestering sites on albumin.
  • IMA cannot sequester metals to the same degree as non-IMA albumin, therefore the resulting current in a sample with a relatively large amount of modified albumin (an ischemic sample) will be different than a sample with very little modified albumin (a normal sample).
  • the type of reaction taking place will determine the type of current flow. For example, if an oxidizing potential is applied, the analyte present in the reaction is being forced from M + ⁇ M 2+ +e ⁇ , rendering electron(s) free for current flow. The converse is true for a reducing potential is applied as the reaction is now being forced from M + +e ⁇ ⁇ M(s), where the system must provide electrons. Therefore, the increased free metal results in a negative current flow within the system.
  • the working electrode measures a net current flow regardless of the direction in which current flows. This system is sensitive enough such that only small variations in the analyte will result in significant changes in net current flow.
  • An amplification system as described herein may or may not be incorporated into the system to increase sensitivity.
  • Potential difference measurements can also be used to measure the amount of IMA in a sample. Measurements can be made by placing both the reference electrode and the ion selective electrode (specific for the analyte being measured) or a combination electrode (also specific to the analyte being measured) into the sample. A potential measurement of the sample is made based on the amount of analyte that can be measured by the ion selective electrode and the reference electrode. The analyte in the proper ratio with albumin can then be added. The potential difference between the ion selective electrode and the reference electrode will change depending on the amount of analyte left after the analyte is sequestered by albumin and when sufficient migration of the analyte to the ion selective electrode has taken place.
  • the electrochemical system measures current changes.
  • a general electrochemical system as shown in FIG. 1 , requires two or three electrodes, a working electrode 2 , reference electrode 4 and optional auxiliary electrode 6 , in contact with each other via a solution in a vessel 8 .
  • the electrodes are connected to a potentiostat 10 that applies a potential and measures a current which is transferred as a signal to an appropriate output device such as a computer 12 or strip chart recorder.
  • a potentiostat consists of two electrodes in a solution, one of which is the reference electrode against which all voltage measurements are referenced.
  • reference electrodes only act properly as a reference when there is no current passing through them. Therefore, depending on the amount of current being produced it may be necessary to have an additional electrode, an auxiliary electrode, through which current can pass, but which is kept at the same voltage as the reference electrode.
  • FIG. 2 The basic components of a functioning potentiostat are shown in FIG. 2 . Three electrodes, the reference 4 , working 2 , and auxiliary 6 electrodes, are placed in a solution containing the ionic species to be measured. A current is passed between the auxiliary electrode 6 and the working electrode 2 .
  • the voltage between the working electrode 2 and the reference electrode 4 is measured, and the current adjusted until the voltage is zero. At this point, the voltage at the working electrode 2 is exactly the same as the voltage at the reference electrode 4 , but no current is passing through the reference electrode 4 .
  • the precise details of a working potentiostat are well known to those skilled in the art.
  • Redox reactions involve the transfer of electron(s).
  • An oxidized species is one that has given up electron(s) to another species.
  • the species that accepts the electron(s) is then said to be a reduced species.
  • the direction in which these electrons flow is driven by a standard redox potential (designated by E°).
  • Redox reactions can be measured in complex matrices, i.e., mixtures or solutions of many materials.
  • Systems for measurement of these reactions require that a potential can be applied to the system relative to a reference electrode.
  • the system requires a potentiostat capable of applying the appropriate potential, measuring the current within working range, and an offset capable of removing background current; a working electrode; a reference electrode and possibly the use of an auxiliary electrode.
  • the working electrode is the site at which the reaction of interest (i.e. the transfer of electrons) occurs.
  • the working electrode can be any solid-state electrode including, but not limited to, platinum, carbon, graphite, gold and mercury/metal amalgam. These solid-state electrodes may or may not be required to be separated from matrix proteins to prevent protein build up on the electrode surface. Polymer coatings or some other filter or barrier separation may be used. Polymer coatings may include, but are not limited to, perfluorosulfonate (NafionTM) and cellulose acetate.
  • the working electrode may also be a semi-solid state or liquid electrode including, but not limited to, carbon paste, mercury film and mercury drop electrodes.
  • the reference electrode provides a relative point for the working electrode to maintain the desired potential.
  • Reference electrodes can be made from silver wire, silver/silver chloride, platinum, carbon, graphite, gold as well as any other solid-state, semi-solid state, and liquid electrode.
  • auxiliary electrode acts to maintain a zero current (preventing current build up) between the reference electrode and the working electrode such that the only current being measured is resulting from the analyte.
  • Auxiliary electrodes may be any of the solid-state electrodes indicated above.
  • Working electrodes may be used as a substrate onto which a species is adsorbed via a redox reaction and then removed and measured with a second redox reaction (see Joseph Wang, Analytical Electrochemistry, 2 nd edition, John Wiley and Sons, 2002). Such techniques are commonly referred to as adsorption or stripping voltammetry.
  • Example 1 describes and FIG. 3 shows the results of such a technique. It was found that increasing concentrations of cobalt added to the sample resulted in corresponding increases in measured current, demonstrating that the current being generated is in fact cobalt dependent.
  • Complexing agents may be added to systems to enhance the signal response of a non-sequestered analyte.
  • Example 2 describes and FIG. 4 illustrates that addition of an excess amount of complexing agent to samples containing increasing concentrations of cobalt ion can enhance the measured current associated with the non-sequestered metal ion.
  • the complexing agent forms a complex with the metal ion, and this complex is measurable in the form of current. Control experiments in which only Co ion or complexing agent was added did not produce a current at the same potentials.
  • the current-generating potential applied to detect the complex of metal ion and complexing agent is typically different from the current-generating potential applied to detect free metal ion in the sample.
  • Direct measurement of cobalt in a biological sample can be made using amperometric or scanning voltammetric techniques as described in Examples 3 and 4 and as illustrated in FIGS. 5 and 6 . Such methods can be used to generate standard curves useful in diagnosis of clinical samples.
  • Ion selective electrodes are commonly used to measure these types of potential differences. Ion selective electrodes selectively measure activity of a species on the inner compartment of the electrode (on one side of the membrane) relative to the outer compartment (the outside of the membrane, or the bulk solution). The differences in activity on either side of the membrane lead to an electric potential difference across the membrane. Ion(s), for which the membrane is selective, diffuse across the membrane based on their concentration gradient. The side of the membrane with low activity will eventually build up charge in common (+/ ⁇ ) with the migrating ion preventing further ion migration at which time a constant potential between the two compartments is reached.
  • a 1 the activity of the ion in one compartment
  • a 2 the activity of the ion in the second compartment
  • ion selective electrodes There are four basic types of ion selective electrodes: glass membrane electrodes, solid-state electrodes, liquid-based electrodes, and compound electrodes.
  • Glass membrane electrodes are typically used for monovalent cations.
  • the most common example is a pH meter that is selective for H+, although different glass compositions are selective for different monovalent cations.
  • Solid-state electrodes use an inorganic salt as the membrane in which the ion of interest can be captured in a pocket in the crystal lattice. The ions can then migrate through the crystal lattice by moving from one adjacent pocket to the next.
  • a common example is the fluoride electrode.
  • Liquid-based electrodes use a hydrophobic polymer membrane saturated with a hydrophobic liquid ion exchanger.
  • the hydrophobic polymer membrane may have an ion “transporter” dissolved into it.
  • An example would be apo-cyanocobolamine dissolved into an organic solvent which would be a selective ion exchanger for cobalt in a hydrophobic membrane.
  • Ion selective electrodes can be scaled down to microelectrodes with tip diameters as small as 10 ⁇ m allowing for very small volume measurements. Ion selective electrodes respond only to the uncomplexed ion, and can be “fine tuned” for selectivity relative to other (potentially interfering) ions by changing parameters discussed above. If the ionic strength of the solution remains constant during a measurement then the activity coefficient remains constant as well, and the measured activity of the ion is equivalent to the concentration of the ion.
  • the device illustrated herein in FIG. 10 can be used in either the current or the potential difference embodiments of the invention.
  • the device consists of an electronics module of suitable size to be held in the hand. It is housed in a rugged water resistant plastic material suitable for hospital use where it may be in contact with body fluids such as blood, and therefore must be easily cleanable.
  • the housing 14 contains the read electronics, power source (e.g., battery), and other components required to perform the measurement operations.
  • function keys 16 and 20 On the housing are one or more function keys 16 and 20 that can be used by the operator for initiating the measurement operation, altering parameters, and entering data such as patient ID.
  • FIG. 10 shows three function keys, but there could be any number from zero to a full keyboard to allow text input.
  • a feature that may also be available on the housing is a mechanism for communication with a laboratory information system (mechanism not shown).
  • a laboratory information system (mechanism not shown).
  • this could be a direct electrical connection (e.g., RS232 serial, and 100BaseT Ethernet communications), an infrared link (transmission via infrared light pulses) or a wireless link (radio transmitter/receiver).
  • a display 18 which is used for displaying results of the potential difference or current measurements, and other communications such as system status, fault conditions, battery low indicator and the like.
  • At one end of the housing is an aperture 30 into which the sample strip is inserted to allow the results to be read.
  • the sample strip is also illustrated in FIG. 10 .
  • the strip 22 may be disposable and preferably is made of an insulating and waterproof material. It contains a sample well or vessel 26 . In the well are electrodes 24 , as described above. Each electrode 24 is connected via its corresponding conductor or trace 32 (which could be a printed conductor or a wire), which is electrically continuous with its corresponding strip contact 28 .
  • a sample of body fluid e.g., blood, urine, saliva
  • the test device is introduced into the aperture 30 in the housing 14 .
  • Electrical contact is made between the strip contacts 28 and contacts in the module aperture (not shown) which are in electrical continuity with the electronics which are also connected to the optional display 18 . Electrical measurements are made on the sample via the electrodes 24 . After the display 18 indicates the results of the measurement sequence and/or the results of the test, the disposable test device may be removed from the module and discarded.
  • the subject methods can be used to detect exercise-induced ischemia.
  • Levels of ischemia in patients at rest and during exercise can be compared to assess patients with known or suspected ischemic conditions. Initially, a patient sample may be obtained and analyzed; then the patient undergoes a treadmill test or other exercise; another patient sample may then be obtained and analyzed; and the results of the tests are compared. These steps may be repeated at additional times (e.g., 3 months, 6 months or 1 year later) for further assessment.
  • This application is useful in assessing the patency of an in-situ coronary stent or the efficacy of a percutaneous cardiac intervention (e.g., an angioplasty procedure).
  • the subject method can also be used to detect the existence of ischemia provoked by exercise in an otherwise asymptomatic patient (silent ischemia). Again, the a sample is taken from the patient, the patient is subject to a treadmill test or the like, and a second sample is obtained. Detection of ischemia in an otherwise asymptomatic patient can reduce the severity of coronary artery disease or other ischemic conditions. Silent ischemia may also be detected with a single sample, i.e., not in the context of an exercise-induction test.
  • the subject methods can be used as a method for ruling out the existence of ischemia in a patient, particularly in patients that possess one or more cardiac risk factors.
  • risk factors include: age greater than 50, history of smoking, diabetes mellitus, obesity, high blood pressure, high cholesterol, and strong family history of cardiac disease.
  • Ruling out an ischemic event is important in proper allocation of medical resources: patients who are known to not be experiencing an ischemic event need not receive the full gamut of tests and treatment reserved for those patients who are in fact experiencing acute coronary syndrome.
  • an initial sample is obtained from the patient, who is then subjected to an exercise treadmill test or other exercise, followed by the taking of a second sample. Comparison of the test results will reveal whether the ischemic event is induced only under the elevated metabolic conditions of exercise. Additionally, ruling out an ischemic event may also be done with a single patient sample, i.e., not in the context of the exercise-induction test.
  • the subject methods can be used to evaluate a patient presenting with angina or angina-like symptoms to detect the occurrence of a myocardial infarction.
  • the results of the subject methods and the results of an ECG are compared for consistency in indicating ischemia and myocardial infarction.
  • the subject methods can be used in the assessment of the efficacy of a percutaneous cardiac intervention such as an angioplasty procedure.
  • a percutaneous cardiac intervention such as an angioplasty procedure.
  • Patient samples are taken before and at predetermined times after the procedure to assess the patient's ischemic status.
  • the subject methods can be used to supplement ECG results so as to determine the occurrence or non-occurrence of an ischemic event.
  • This application may find particular use in patients undergoing surgery.
  • the subject methods can also be used to detect the occurrence of a temporally-limited ischemic event in a patient
  • Temporally-limited ischemia includes ischemic events associated with, e.g., exercise or drug use.
  • the subject method can also be used to detect an ischemic event in a patient suffering from stroke-like signs. Use of the subject methods in this situation permits the distinction between an ischemic stroke and a hemorrhagic stroke.
  • the subject methods can be used to assess the efficacy of thrombolytic or other drug therapy such as drugs to attenuate an ischemic event by conditioning ischemic myocardium.
  • Patient samples are obtained before and at predetermined times after the drug therapy to detect any reduction in ischemic events due to the therapy.
  • the subject methods can be used for the detection of placental insufficiency in a pregnant woman.
  • the sample is typically taken from the mother; the presence of IMA can be indicative of an ischemic condition in the placenta
  • Adsorption or stripping voltammetry was used to correlate increasing cobalt concentration in a sample with generated current.
  • Cobalt was added to a biological fluid and allowed time to bind to non-modified albumin. The kinetics of cobalt binding to albumin were tested previously and showed the binding reaction was faster than 1 minute.
  • a series of potentials were applied, in this case in the form of a cyclic voltammogram, although potentials may also be applied step-wise, resulting in a redox reaction of the available cobalt, thus causing cobalt to adsorb to the working electrode.
  • a second cobalt redox reaction measured the amount of cobalt absorbed to the electrode in the form of current as a range of potentials is scanned.
  • Cobalt dependent current peaks generated in this fashion are shown over a concentration range of 56–174 mM cobalt in FIG. 3 .
  • a change in current consistent with a change in cobalt concentration was observed demonstrating that the current being generated is in fact cobalt dependent.
  • the complexing agent DMG was added to samples to enhance the signal response of cobalt ion. Increasing concentrations of cobalt were added to a biological sample and allowed to bind to non-modified albumin. A constant concentration of DMG was then added in excess of the cobalt concentration to the sample and voltammetry techniques were used to detect the Co(DMG) 2 complex in the form of current. In this case, a cobalt concentration range of 0.094–1.780 mM was used and showed the appropriate change in current value relative the change in cobalt concentration. In control experiments, cobalt and DMG were added separately and showed no current response at the appropriate potentials, indicating that the signal generated is dependent on the formation of Co(DMG) 2 complex and is therefore proportional to the amount of available cobalt present in the system.
  • Cobalt may also be measured directly in a biological sample using cyclic voltammetry or similar scanning potential techniques such as step voltammetry. Cobalt, excess to the amount of albumin present in the sample, was added and allowed to bind to non-modified albumin. The appropriate potential range was then scanned such that a cobalt dependent current was measured.
  • FIG. 6 shows the resulting plot of increasing cobalt current response over an increasing range of cobalt concentrations (0–1200 ⁇ M), indicating that the current response is cobalt dependent and that a relative concentration range can easily be detected.
  • FIG. 7 shows the expected decrease in the cobalt current response with increasing concentrations of albumin, which is indicative that albumin sequesters cobalt from detection by electrochemical methods.
  • One of the proposed ischemia mechanisms involves the binding of copper to the N-terminus of albumin during an ischemic event.
  • Ischemic samples were simulated by adding increasing concentrations of copper to a normal sample, a method that has shown an average increase in ACB value of 60% at 1 mM copper.
  • Cobalt was then added to the sample and the appropriate potential range was scanned to produce a cobalt dependent current peak resulting from the redox reaction of cobalt.
  • FIG. 8 shows the response in the electrochemical system under these conditions. As copper concentrations increase and the copper becomes bound to the sequestering sites on albumin, there are fewer sites available to sequester cobalt and therefore there is an increase in the cobalt current response.
  • the increase in electrochemical output signal (current) is approximately 30%.
  • the increased current is due to cobalt because there will be no competition in binding to the primary site as copper has a higher affinity for albumin than does cobalt (10 16 and 10 4 respectively).
  • the redox potential for copper does not overlap with the redox potential of cobalt.
  • Ion selective electrodes are commonly used in the clinical setting to measure electrolytes (e.g., Na+ and K+) in biological samples. Valinomycin is often used to make a membrane selective for potassium (Cattrall, R. wt al. (1974) Anal. Chem. 46(14):2223–6). ISE have also been shown to be useful in measuring copper binding to serum albumins (Mohanakrishnan, P. et al. (1982) J. Pharm. Sci. 71(10):1180–2), thus demonstrating the ability of ISE to measure free metal ions in a complex sample matrix. Altura et al. (1996) Scand. J. Clin. Lab. Invest. Suppl. 224:211–34, demonstrated that there was no observable difference between ISE signals obtained from plasma and serum samples when measuring magnesium concentrations.
  • an existing method for detecting albumin-cobalt binding involves the detection of free cobalt in a patient sample using a calorimetric reaction.
  • the subject example demonstrates how free cobalt can be detected in biological samples using an ion selective electrode and then correlating the results to the current ACB Test format.
  • a standard curve is generated such that the resulting potential difference between the working and reference electrodes can be translated into free cobalt concentrations.
  • a sample consisting of 6 g/dL IgG in 0.9% NaCl is used to simulate the expected sample matrix conditions in which there is no capacity to sequester added cobalt.
  • the working electrode and the reference electrode are both placed in contact with the sample.
  • Varying concentrations (5, 10, 15, 20 and 25 mM) of cobalt chloride solutions are added to the sample and the resulting potentials are recorded.
  • the plotted results of potential versus cobalt concentration should ideally be linear or a non linear function can be used to fit the curve.
  • the line or curve fitting equation for the plot is recorded such that subsequent experimental measurements of free cobalt can be quantified.
  • Equipment and reagents should be recalibrated before sample testing if the working electrode has not been used for 24 hours or if there is a reagent change.
  • the selectivity of the ion selective electrode is determined by the selectivity coefficient.
  • Y any other species of size, charge or properties similar to cobalt that the ISE may respond to.
  • Y may also include free metal ions of high concentration found in the sample type to be tested.
  • the selectivity coefficient should be very small (K ⁇ 1) indicating that the electrode is highly selective for cobalt.
  • a sample consisting of 6 g/dL IgG in 0.9% NaCl is used to mimic the physiological matrix. Varying concentrations (5, 10, 15, 20, and 25 mM) of FeCl 2 are added to the sample and the resulting potentials between the working electrode and the reference electrode at each concentration are recorded.
  • the selectivity coefficient for Co 2+ over Fe 2+ is calculated by taking the potential response from 5 mM Fe 2+ and dividing by the potential response from 5 mM Co 2+ . This calculation is continued for all concentrations of Fe 2+ tested. All selectivity coefficients should be very small.
  • the working and reference electrodes are placed in the biological sample (e.g., plasma, serum, whole blood). No potential difference or very little potential difference should be measured at this time as the working electrode can only measure free cobalt of which there should very little or none present in the sample. If a signal is measured it is assumed to be background noise and can be subtracted from the final measurement. 25 mM cobalt chloride solution is added to the sample and the stabilized potential difference between the working electrode and reference electrode is measured. When the signal has stabilized, all the albumin sites capable of binding and sequestering cobalt are occupied and the signal (in millivolts, mV) is produced from available non sequestered cobalt in the sample.
  • the biological sample e.g., plasma, serum, whole blood.
  • Available non sequestered cobalt in the sample can then be quantified using the stabilized potential measurement and referenced to the appropriate calibration curve.
  • the existing albumin-cobalt binding (ACB) Test set forth in PCT/US99/22905 measures IMA in samples by calorimetrically measuring available non-sequestered cobalt.
  • a correlation between the ACB method and the ISE method can be established by testing samples using both methods.
  • a plot is made where mV results from the ISE method are plotted on the y-axis and the ACB (U/mL) results are plotted on the x-axis.
  • the correlation coefficient for the resultant line is an indication of the relative performance of the ISE method relative to the ACB Test.
  • a bias plot should be generated by calculating the percent difference between the corresponding individual samples as measured by both methods to determine the accuracy of the new method.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
US10/304,610 2001-11-26 2002-11-26 Electrochemical detection of ischemia Expired - Lifetime US7063782B2 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US10/304,610 US7063782B2 (en) 2001-11-26 2002-11-26 Electrochemical detection of ischemia
US11/415,929 US20060191788A1 (en) 2001-11-26 2006-05-01 Electrochemical detection of ischemia

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US33367701P 2001-11-26 2001-11-26
US10/304,610 US7063782B2 (en) 2001-11-26 2002-11-26 Electrochemical detection of ischemia

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US11/415,929 Division US20060191788A1 (en) 2001-11-26 2006-05-01 Electrochemical detection of ischemia

Publications (2)

Publication Number Publication Date
US20030132125A1 US20030132125A1 (en) 2003-07-17
US7063782B2 true US7063782B2 (en) 2006-06-20

Family

ID=23303798

Family Applications (2)

Application Number Title Priority Date Filing Date
US10/304,610 Expired - Lifetime US7063782B2 (en) 2001-11-26 2002-11-26 Electrochemical detection of ischemia
US11/415,929 Abandoned US20060191788A1 (en) 2001-11-26 2006-05-01 Electrochemical detection of ischemia

Family Applications After (1)

Application Number Title Priority Date Filing Date
US11/415,929 Abandoned US20060191788A1 (en) 2001-11-26 2006-05-01 Electrochemical detection of ischemia

Country Status (6)

Country Link
US (2) US7063782B2 (fr)
EP (1) EP1459059A4 (fr)
JP (1) JP2005510712A (fr)
AU (1) AU2002352959B2 (fr)
CA (1) CA2468236A1 (fr)
WO (1) WO2003046538A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080302659A1 (en) * 2007-06-07 2008-12-11 Microchips, Inc. Electrochemical biosensors and arrays
US20090004686A1 (en) * 2007-05-18 2009-01-01 Institute For Molecular Medicine, Inc. Measurement and uses of oxidative stress
WO2009020860A1 (fr) 2007-08-03 2009-02-12 Virginia Commonwealth University Procédé pour diagnostiquer l'ischémie cardiaque aiguë
US20100267074A1 (en) * 2007-05-18 2010-10-21 Institute For Molecular Medicine, Inc. Measurement and uses of oxidative stress
US8317997B2 (en) 2011-02-28 2012-11-27 Institute For Molecular Medicine, Inc. Method and apparatus for measuring oxidation-reduction potential
US8514067B2 (en) 2011-08-16 2013-08-20 Elwha Llc Systematic distillation of status data relating to regimen compliance
US9360446B2 (en) 2012-10-23 2016-06-07 Aytu Bioscience, Inc. Methods and systems for measuring and using the oxidation-reduction potential of a biological sample
US9372167B2 (en) 2012-04-19 2016-06-21 Aytu Bioscience, Inc. Oxidation-reduction potential test device including a multiple layer gel
US9626650B2 (en) 2011-04-14 2017-04-18 Elwha Llc Cost-effective resource apportionment technologies suitable for facilitating therapies
WO2017116358A1 (fr) 2015-12-28 2017-07-06 Oran Ismail Procédé et appareil de détermination de marqueurs d'acides gras par mesure d'impédance électrique
US10445846B2 (en) 2011-04-14 2019-10-15 Elwha Llc Cost-effective resource apportionment technologies suitable for facilitating therapies

Families Citing this family (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7964146B2 (en) 2004-05-30 2011-06-21 Agamatrix, Inc. Measuring device and methods for use therewith
EP1756558A1 (fr) * 2004-06-14 2007-02-28 Oxford Biosensors Limited Procede de fabrication d'electrode en microbande
GB0414550D0 (en) * 2004-06-29 2004-08-04 Oxford Biosensors Ltd Electrochemical sensing method
GB0414546D0 (en) * 2004-06-29 2004-08-04 Oxford Biosensors Ltd Electrode for electrochemical sensor
GB0414548D0 (en) * 2004-06-29 2004-08-04 Oxford Biosensors Ltd Electrode preconditioning
GB0414551D0 (en) * 2004-06-29 2004-08-04 Oxford Biosensors Ltd Electrochemical sensor
US7373195B2 (en) * 2004-07-30 2008-05-13 Medtronic, Inc. Ion sensor for long term use in complex medium
GB2417323A (en) * 2004-08-17 2006-02-22 Oxford Biosensors Ltd A method of operating an electrochemical sensor by applying a time variable potential between the electrodes.
WO2006079032A2 (fr) * 2005-01-21 2006-07-27 Ischemia Technologies, Inc. Cibles pour la detection d'ischemie
KR100733414B1 (ko) * 2005-04-30 2007-06-29 주식회사 하이닉스반도체 내부전원 생성장치
GB2464222B (en) * 2006-02-15 2010-07-07 Inverness Medical Switzerland Analysis method
GB0617581D0 (en) * 2006-09-07 2006-10-18 Ucl Business Plc Biomarkers for assessing liver function
WO2008046086A2 (fr) * 2006-10-12 2008-04-17 Pipex, Inc. Instruments pour la détection sélective et directe de métaux libres dans des fluides, et procédés pour diagnostiquer des maladies liées au métal et pour déterminer des régimes de dosage pharmacologique
WO2008075193A2 (fr) * 2006-12-19 2008-06-26 Inverness Medical Switzerland Gmbh Dispositifs et procédés de dosage
US20100015728A1 (en) * 2006-12-19 2010-01-21 Iverness Medical Switzerland Gmbh Assay Device and Method
US20080176702A1 (en) * 2007-01-19 2008-07-24 Showalter Dan J Torque vectoring system
ES1071520Y (es) * 2009-12-14 2010-06-04 Univ Illes Balears Dispositivo portatil de analisis del ph de la orina
EP2742348A4 (fr) * 2011-08-12 2015-07-22 Alfred Health Procédé pour le diagnostic, le pronostic ou le traitement du syndrome coronarien aigu (sca) comprenant la mesure de la concentration plasmatique de facteur inhibiteur de migration des macrophages (mif)
KR101552385B1 (ko) * 2013-03-18 2015-09-10 이화여자대학교 산학협력단 심근허혈에 의한 알부민을 측정하기 위한 신규한 알부민―구리 결합 측정법
WO2016038505A2 (fr) 2014-09-08 2016-03-17 Indian Institute Of Science Biocapteur électrochimique et procédé de détection de l'albumine et de ses complexes
US11867654B2 (en) * 2015-05-12 2024-01-09 Indian Institute Of Science Device and method for detecting creatinine and albumin to creatinine ratio
EP4265185A1 (fr) * 2015-12-22 2023-10-25 Radiometer Medical ApS Procédé de détection de présence ou d'absence d'un caillot dans un analyseur d'échantillons liquides
WO2024063638A1 (fr) * 2022-09-23 2024-03-28 Universiti Kebangsaan Malaysia Dispositif de détection d'albumine à partir de salive

Citations (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5110746A (en) 1987-04-22 1992-05-05 Pharmacia Diagnostics Inc. Method for the determination of albumin in biological fluids
US5141855A (en) 1986-07-28 1992-08-25 Eastman Kodak Company Signal amplifying cobalt (III) redox reagents and methods for the determination of analytes in aqueous fluids
US5227307A (en) 1991-07-26 1993-07-13 Diagnostic Markers, Inc. Test for the rapid evaluation of ischemic state
US5273639A (en) * 1988-03-31 1993-12-28 Agency Of Industrial Science & Technology Probe electrode
US5288636A (en) 1989-12-15 1994-02-22 Boehringer Mannheim Corporation Enzyme electrode system
US5366609A (en) 1993-06-08 1994-11-22 Boehringer Mannheim Corporation Biosensing meter with pluggable memory key
US5368707A (en) 1992-01-15 1994-11-29 Andcare, Inc. Convenient determination of trace lead in whole blood and other fluids
US5460972A (en) 1991-04-08 1995-10-24 Research Foundation Of The State University Of New York Ionized magnesium2+ concentrations in biological samples
US5653864A (en) 1994-06-30 1997-08-05 Nok Corporation Protein biosensor and method for protein measurement with the same
WO2000020454A1 (fr) 1998-10-02 2000-04-13 Diagnostic Markers, Inc. Procedes et substances pour detecter et mesurer les lesions provoquees par des radicaux libres
WO2000020840A1 (fr) 1998-10-02 2000-04-13 Ischemia Technologies, Inc. Epreuves permettant une evaluation rapide d'etats ischemiques et necessaires a cet effet
US6143164A (en) 1997-02-06 2000-11-07 E. Heller & Company Small volume in vitro analyte sensor
WO2000078210A1 (fr) 1999-06-17 2000-12-28 Minimed Inc. Systeme moniteur de caracteristiques utilise avec un capteur d'analyte
US6461875B1 (en) 1998-10-02 2002-10-08 Ischemia Technologies, Inc. Test for rapid evaluation of ischemic states and kit
US6475743B1 (en) 1998-10-02 2002-11-05 Ischemia Technologies, Inc. Marker useful for detection and measurement of free radical damage and method
WO2002089656A2 (fr) 2001-05-04 2002-11-14 Ischemia Technologies, Inc. Ameliorations apportees au diagnostic de l'infarctus aigu du myocarde et d'autres conditions cliniques
WO2002096266A2 (fr) 2001-05-30 2002-12-05 Ischemia Technology, Inc. Methode et systeme d'analyse optique permettant de detecter un etat pathologique
US6492179B1 (en) 1998-10-02 2002-12-10 Ischemia Techologies, Inc. Test for rapid evaluation of ischemic states and kit
WO2003025571A1 (fr) 2001-09-14 2003-03-27 Alan Kleinfeld Marqueurs diagnostiques de l'ischemie
WO2004032711A2 (fr) 2002-10-09 2004-04-22 Dmi Biosciences, Inc. Diagnostic et surveillance d'une inflammation, d'une ischemie et d'une appendicite
WO2004054431A2 (fr) 2002-12-13 2004-07-01 Ischemia Technologies, Inc. Tests d'evaluation rapide d'etats ischemiques et necessaires
US6770487B2 (en) 2001-05-01 2004-08-03 Ischemia Technologies, Inc. Bar code readable diagnostic strip test

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5110745A (en) * 1989-06-01 1992-05-05 The Trustees Of The University Of Pennsylvania Methods of detecting glycated proteins
US5508171A (en) * 1989-12-15 1996-04-16 Boehringer Mannheim Corporation Assay method with enzyme electrode system
US5246858A (en) * 1991-02-27 1993-09-21 Boehringer Mannheim Corporation Apparatus and method for analyzing a body fluid
DE69427911T2 (de) * 1993-06-21 2002-04-04 Roche Diagnostics Corp., Indianapolis Stabilisator für diagnostische reagentien
US5762770A (en) * 1994-02-21 1998-06-09 Boehringer Mannheim Corporation Electrochemical biosensor test strip
US5997817A (en) * 1997-12-05 1999-12-07 Roche Diagnostics Corporation Electrochemical biosensor test strip
US6413213B1 (en) * 2000-04-18 2002-07-02 Roche Diagnostics Corporation Subscription based monitoring system and method

Patent Citations (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5141855A (en) 1986-07-28 1992-08-25 Eastman Kodak Company Signal amplifying cobalt (III) redox reagents and methods for the determination of analytes in aqueous fluids
US5110746A (en) 1987-04-22 1992-05-05 Pharmacia Diagnostics Inc. Method for the determination of albumin in biological fluids
US5273639A (en) * 1988-03-31 1993-12-28 Agency Of Industrial Science & Technology Probe electrode
US5288636A (en) 1989-12-15 1994-02-22 Boehringer Mannheim Corporation Enzyme electrode system
US5460972A (en) 1991-04-08 1995-10-24 Research Foundation Of The State University Of New York Ionized magnesium2+ concentrations in biological samples
US5227307A (en) 1991-07-26 1993-07-13 Diagnostic Markers, Inc. Test for the rapid evaluation of ischemic state
US5290519A (en) 1991-07-26 1994-03-01 Diagnostic Markers, Inc. Test for the rapid evaluation of ischemic states and kit
US5368707A (en) 1992-01-15 1994-11-29 Andcare, Inc. Convenient determination of trace lead in whole blood and other fluids
US5366609A (en) 1993-06-08 1994-11-22 Boehringer Mannheim Corporation Biosensing meter with pluggable memory key
US5653864A (en) 1994-06-30 1997-08-05 Nok Corporation Protein biosensor and method for protein measurement with the same
US6143164A (en) 1997-02-06 2000-11-07 E. Heller & Company Small volume in vitro analyte sensor
WO2000020454A1 (fr) 1998-10-02 2000-04-13 Diagnostic Markers, Inc. Procedes et substances pour detecter et mesurer les lesions provoquees par des radicaux libres
US6461875B1 (en) 1998-10-02 2002-10-08 Ischemia Technologies, Inc. Test for rapid evaluation of ischemic states and kit
US6475743B1 (en) 1998-10-02 2002-11-05 Ischemia Technologies, Inc. Marker useful for detection and measurement of free radical damage and method
WO2000020840A1 (fr) 1998-10-02 2000-04-13 Ischemia Technologies, Inc. Epreuves permettant une evaluation rapide d'etats ischemiques et necessaires a cet effet
US6492179B1 (en) 1998-10-02 2002-12-10 Ischemia Techologies, Inc. Test for rapid evaluation of ischemic states and kit
WO2000078210A1 (fr) 1999-06-17 2000-12-28 Minimed Inc. Systeme moniteur de caracteristiques utilise avec un capteur d'analyte
US6770487B2 (en) 2001-05-01 2004-08-03 Ischemia Technologies, Inc. Bar code readable diagnostic strip test
WO2002089656A2 (fr) 2001-05-04 2002-11-14 Ischemia Technologies, Inc. Ameliorations apportees au diagnostic de l'infarctus aigu du myocarde et d'autres conditions cliniques
WO2002096266A2 (fr) 2001-05-30 2002-12-05 Ischemia Technology, Inc. Methode et systeme d'analyse optique permettant de detecter un etat pathologique
WO2003025571A1 (fr) 2001-09-14 2003-03-27 Alan Kleinfeld Marqueurs diagnostiques de l'ischemie
WO2004032711A2 (fr) 2002-10-09 2004-04-22 Dmi Biosciences, Inc. Diagnostic et surveillance d'une inflammation, d'une ischemie et d'une appendicite
US20040175754A1 (en) 2002-10-09 2004-09-09 David Bar-Or Diagnosis and monitoring of inflammation, ischemia and appendicitis
WO2004054431A2 (fr) 2002-12-13 2004-07-01 Ischemia Technologies, Inc. Tests d'evaluation rapide d'etats ischemiques et necessaires

Non-Patent Citations (24)

* Cited by examiner, † Cited by third party
Title
Altura et al. (1996) Scand. J. Clin. Lab. Invest. Suppl. 224:211.
Ashtamkar, S. M.; Thakkar, N. V.; "Preparation and study of an epoxy resin based cobalt (KK) ion selective electrode using a cobalt (II) complex," Jan. 2002, J. Indian Chem. Soc., vol. 79, 90-91. *
Bar-Or et al. (2000) J. Emerg. Med. 19:311-315.
Bar-Or et al. (2001) Eur. J. Biochem. 268(1):42-47.
Berenshtein et al. (1997) J Mol Cell Cardiol. 29(11):3025-3034.
Bradshaw et al. (1968) J Biol. Chem. 243:3817-3825.
Bradshaw et al. (1969) J. Biol. Chem. 244:5582-5589.
Cai et al. (2001) J. Biochem. Biophys. Methods 47(3):209-219.
Cattrall et al. (1974) Anal. Chem. 46(14):2223.
Christenson et al. (2001) Clin. Chem. 47:464-470.
Gryzunov et al. (2003) Arch Biochem. Biphys. 413(1):53-66.
John et al. (1991) Analytica Chimica Acta 249:381-385.
Kaku et al. (1993) Analytica Chimica Acta 272:213-220.
Kolthoff et al. (1977) Anal. Chem. 49(13):2108-2109.
Lakusta et al. (1979) J. Inorg. Biochem. 11:303.
Laussac et al. (1984) Biochem. 23:2832.
Lee et al. (1980) Am. J. Med. Technol. 46(12):853-7.
Masuoka et al. (1993) J. Biol. Chem. 268:21533.
Mohanakrishnan et al. (1982) J. Pharm. Sci. 71(10):1180.
Predki et al. (1992) Biochem. J. 287:211-215.
Sadler et al. (1994) Eur. J. Biochem. 220:193.
Wang et al. (1998) Anal. Chem. 70(9):1682-1685.
Wang et al. (2000) Anal. Chem. 72(14):3218-22.
Wang, Analytical Electrochemistry, 2<SUP>nd </SUP>edition, John Wiley and Sons, 2002 p. 74.

Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8709709B2 (en) 2007-05-18 2014-04-29 Luoxis Diagnostics, Inc. Measurement and uses of oxidative status
US20090004686A1 (en) * 2007-05-18 2009-01-01 Institute For Molecular Medicine, Inc. Measurement and uses of oxidative stress
US20100267074A1 (en) * 2007-05-18 2010-10-21 Institute For Molecular Medicine, Inc. Measurement and uses of oxidative stress
US10036723B2 (en) 2007-05-18 2018-07-31 Aytu Bioscience, Inc. Measurement and uses of oxidative status
US8512548B2 (en) 2007-05-18 2013-08-20 Luoxis Diagnostics, Inc. Measurement and uses of oxidative status
US9423372B2 (en) 2007-05-18 2016-08-23 Aytu Bioscience, Inc. Measurement and uses of oxidative status
US9383331B2 (en) 2007-05-18 2016-07-05 Aytu Bioscience, Inc. Measurement and uses of oxidative status
US9063070B2 (en) 2007-05-18 2015-06-23 Luoxis Diagnostics, Inc. Measurement and uses of oxidative status
US20080302659A1 (en) * 2007-06-07 2008-12-11 Microchips, Inc. Electrochemical biosensors and arrays
US8649840B2 (en) * 2007-06-07 2014-02-11 Microchips, Inc. Electrochemical biosensors and arrays
WO2009020860A1 (fr) 2007-08-03 2009-02-12 Virginia Commonwealth University Procédé pour diagnostiquer l'ischémie cardiaque aiguë
US9528959B2 (en) 2011-02-28 2016-12-27 Aytu Bioscience, Inc. Method and apparatus for measuring oxidation-reduction potential
US8329012B2 (en) 2011-02-28 2012-12-11 Institute For Molecular Medicine, Inc. Method and apparatus for measuring oxidation-reduction potential
US9034159B2 (en) 2011-02-28 2015-05-19 Luoxis Diagnostics, Inc. Method and apparatus for measuring oxidation-reduction potential
US8641888B2 (en) 2011-02-28 2014-02-04 Luoxis Diagnostics, Inc. Method and apparatus for measuring oxidation-reduction potential
US8317997B2 (en) 2011-02-28 2012-11-27 Institute For Molecular Medicine, Inc. Method and apparatus for measuring oxidation-reduction potential
US10853819B2 (en) 2011-04-14 2020-12-01 Elwha Llc Cost-effective resource apportionment technologies suitable for facilitating therapies
US10445846B2 (en) 2011-04-14 2019-10-15 Elwha Llc Cost-effective resource apportionment technologies suitable for facilitating therapies
US9626650B2 (en) 2011-04-14 2017-04-18 Elwha Llc Cost-effective resource apportionment technologies suitable for facilitating therapies
US9770189B2 (en) 2011-08-16 2017-09-26 Elwha Llc Systematic distillation of status data relating to regimen compliance
US8514067B2 (en) 2011-08-16 2013-08-20 Elwha Llc Systematic distillation of status data relating to regimen compliance
US8599009B2 (en) 2011-08-16 2013-12-03 Elwha Llc Systematic distillation of status data relating to regimen compliance
US8723640B2 (en) 2011-08-16 2014-05-13 Elwha Llc Distillation of status data relating to regimen compliance responsive to the presence and absence of wireless signals relating to one or more threshold frequencies
US8816814B2 (en) 2011-08-16 2014-08-26 Elwha Llc Systematic distillation of status data responsive to whether or not a wireless signal has been received and relating to regimen compliance
US9372167B2 (en) 2012-04-19 2016-06-21 Aytu Bioscience, Inc. Oxidation-reduction potential test device including a multiple layer gel
US10281425B2 (en) 2012-04-19 2019-05-07 Aytu Bioscience, Inc. Multiple layer gel
US9410913B2 (en) 2012-10-23 2016-08-09 Aytu Bioscience, Inc. Methods and systems for measuring and using the oxidation-reduction potential of a biological sample
US10184931B2 (en) 2012-10-23 2019-01-22 Aytu Bioscience, Inc. Methods and systems for measuring and using the oxidation-reduction potential of a biological sample
US9360446B2 (en) 2012-10-23 2016-06-07 Aytu Bioscience, Inc. Methods and systems for measuring and using the oxidation-reduction potential of a biological sample
WO2017116358A1 (fr) 2015-12-28 2017-07-06 Oran Ismail Procédé et appareil de détermination de marqueurs d'acides gras par mesure d'impédance électrique

Also Published As

Publication number Publication date
AU2002352959B2 (en) 2008-04-10
EP1459059A4 (fr) 2005-01-19
WO2003046538A1 (fr) 2003-06-05
US20060191788A1 (en) 2006-08-31
AU2002352959A1 (en) 2003-06-10
EP1459059A1 (fr) 2004-09-22
US20030132125A1 (en) 2003-07-17
CA2468236A1 (fr) 2003-06-05
JP2005510712A (ja) 2005-04-21

Similar Documents

Publication Publication Date Title
US7063782B2 (en) Electrochemical detection of ischemia
JP2005510712A5 (fr)
EP2359146A1 (fr) Procédé électrochimique non enzymatique pour la détermination simultanée de l hémoglobine totale et de l hémoglobine glyquée
WO2009125563A1 (fr) Système biocapteur, pointe de détection et procédé de mesure de la concentration d&#39;une substance à analyser dans un échantillon de sang
KR20100008260A (ko) 바이오센서를 이용한 단백질 측정 장치
JPS637344B2 (fr)
JP2018515745A (ja) クレアチニンおよびアルブミン／クレアチニン比を検出するための装置および方法
KR102024995B1 (ko) 혈액 분석 장치 및 이를 이용한 혈액 분석 방법
Brainina et al. Platinum electrode regeneration and quality control method for chronopotentiometric and chronoamperometric determination of antioxidant activity of biological fluids
JP2017530371A (ja) 電気化学的バイオセンサ及びアルブミンとその複合体の検出方法
Amreen et al. Undiluted human whole blood uric acid detection using a graphitized mesoporous carbon modified electrode: A potential tool for clinical point-of-care uric acid diagnosis
KR102102174B1 (ko) 혈액 분석 장치 및 이를 이용한 혈액 분석 방법
US20040099531A1 (en) Methods and apparatus for electrochemically testing samples for constituents
JP7417664B2 (ja) 心臓バイオマーカーのインピーダンス測定による検出のための垂直配向のプラチナワイヤ・アプタセンサアレイの作成およびパラメータ評価
US10670580B2 (en) Quantification of inflammatory molecules in exhaled breath condensate using differential pulse voltammetry on reduced graphene oxide sensor
Lewenstam Clinical analysis of blood gases and electrolytes by ion-selective sensors
US20060042961A1 (en) Potentiometric measurement of chloride concentration in an acidic solution
JP7371219B2 (ja) 分析物測定システムで使用されるバイオセンサの汚染の決定
JP2008505335A (ja) 虚血を検出するための組成物および電気化学センサー
Li et al. Voltammetry in sheep's blood: Membrane-free amperometric measurement of O2 concentration
RU2839135C1 (ru) Биосенсорное аналитическое устройство для количественной оценки содержания этанола в физиологических жидкостях
KR910002647B1 (ko) 단일 결정식 혈액분석방법 및 그 장치
CN108132284A (zh) 一种电化学传感器的测试方法
WO2020250190A1 (fr) Dispositif et procédé de dosage rapide d&#39;ions cuivre et d&#39;autres métaux lourds sur des micro-volumes de sang capillaire humain et d&#39;autres liquides biologiques
KR20210078001A (ko) 현장 자가 진단을 위한 비표지 전기화학센싱 방법

Legal Events

Date Code Title Description
AS Assignment

Owner name: ISCHEMIA TECHNOLOGIES, INC., COLORADO

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WAYMENT, HOLLIE;FAGAN, GARY;CROSBY, PETER A.;AND OTHERS;REEL/FRAME:013505/0641

Effective date: 20030305

AS Assignment

Owner name: SILICON VALLEY BANK, CALIFORNIA

Free format text: SECUIRITY AGREEMENT;ASSIGNOR:ISCHEMIA TECHNOLOGIES, INC.;REEL/FRAME:014007/0147

Effective date: 20030410

AS Assignment

Owner name: ISCHEMIA TECHNOLOGIES, INC., COLORADO

Free format text: RELEASE OF SECURITY INTEREST;ASSIGNOR:SILICON VALLEY BANK;REEL/FRAME:016561/0080

Effective date: 20050316

AS Assignment

Owner name: GENERAL ELECTRIC CAPITAL CORPORATION, AS AGENT, MA

Free format text: SECURITY INTEREST;ASSIGNOR:ISCHEMIA TECHNOLOGIES, INC.;REEL/FRAME:016570/0005

Effective date: 20050316

STCF Information on status: patent grant

Free format text: PATENTED CASE

AS Assignment

Owner name: GENERAL ELECTRIC CAPITAL CORPORATION, AS AGENT,MAR

Free format text: FIRST LIEN INTELLECTUAL PROPERTY SECURITY AGREEMENT;ASSIGNORS:IM US HOLDINGS, LLC, A DELAWARE LIMITED LIABILITY COMPANY;INVERNESS MEDICAL INNOVATIONS, INC., A DELAWARE CORPORATION;ADVANTAGE DIAGNOSTICS CORPORATION, A DELAWARE CORPORATION;AND OTHERS;REEL/FRAME:019477/0268

Effective date: 20070626

Owner name: GENERAL ELECTRIC CAPITAL CORPORATION, AS AGENT, MA

Free format text: FIRST LIEN INTELLECTUAL PROPERTY SECURITY AGREEMENT;ASSIGNORS:IM US HOLDINGS, LLC, A DELAWARE LIMITED LIABILITY COMPANY;INVERNESS MEDICAL INNOVATIONS, INC., A DELAWARE CORPORATION;ADVANTAGE DIAGNOSTICS CORPORATION, A DELAWARE CORPORATION;AND OTHERS;REEL/FRAME:019477/0268

Effective date: 20070626

AS Assignment

Owner name: GENERAL ELECTRIC CAPITAL CORPORATION, AS AGENT,MAR

Free format text: SECOND LIEN INTELLECTUAL PROPERTY SECURITY AGREEMENT;ASSIGNORS:IM US HOLDINGS, LLC, A DELAWARE LIMITED LIABILITY COMPANY;INVERNESS MEDICAL INNOVATIONS, INC., A DELAWARE CORPORATION;ADVANTAGE DIAGNOSTICS CORPORATION, A DELAWARE CORPORATION;AND OTHERS;REEL/FRAME:019477/0808

Effective date: 20070626

Owner name: GENERAL ELECTRIC CAPITAL CORPORATION, AS AGENT, MA

Free format text: SECOND LIEN INTELLECTUAL PROPERTY SECURITY AGREEMENT;ASSIGNORS:IM US HOLDINGS, LLC, A DELAWARE LIMITED LIABILITY COMPANY;INVERNESS MEDICAL INNOVATIONS, INC., A DELAWARE CORPORATION;ADVANTAGE DIAGNOSTICS CORPORATION, A DELAWARE CORPORATION;AND OTHERS;REEL/FRAME:019477/0808

Effective date: 20070626

FPAY Fee payment

Year of fee payment: 4

AS Assignment

Owner name: GENERAL ELECTRIC CAPITAL CORPORATION, MARYLAND

Free format text: SECURITY AGREEMENT;ASSIGNORS:ADVANTAGE DIAGNOSTICS CORPORATION;ALERE MEDICAL INCORPORATED;ALERE SAN DIEGO, INC.;AND OTHERS;REEL/FRAME:026557/0287

Effective date: 20110630

FPAY Fee payment

Year of fee payment: 8

AS Assignment

Owner name: ADVANTAGE DIAGNOSTICS CORPORATION, CALIFORNIA

Free format text: NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT;REEL/FRAME:036011/0581

Effective date: 20150618

Owner name: MATRIA HEALTHCARE, INC., GEORGIA

Free format text: NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT;REEL/FRAME:036011/0581

Effective date: 20150618

Owner name: BINAX, INC., MAINE

Free format text: NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT;REEL/FRAME:036011/0581

Effective date: 20150618

Owner name: CHOLESTECH CORPORATION, CALIFORNIA

Free format text: NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT;REEL/FRAME:036011/0581

Effective date: 20150618

Owner name: ALERE MEDICAL, INC., NEVADA

Free format text: NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT;REEL/FRAME:036011/0581

Effective date: 20150618

Owner name: INVERNESS MEDICAL - BIOSTAR INC., MASSACHUSETTS

Free format text: NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT;REEL/FRAME:036011/0581

Effective date: 20150618

Owner name: GENECARE MEDICAL GENETICS CENTER, INC., NORTH CARO

Free format text: NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT;REEL/FRAME:036011/0581

Effective date: 20150618

Owner name: ISCHEMIA TECHNOLOGIES, INC., MASSACHUSETTS

Free format text: NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT;REEL/FRAME:036011/0581

Effective date: 20150618

Owner name: HEMOSENSE, INC., CALIFORNIA

Free format text: NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT;REEL/FRAME:036011/0581

Effective date: 20150618

Owner name: BIOSITE INCORPORATED, CALIFORNIA

Free format text: NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT;REEL/FRAME:036011/0581

Effective date: 20150618

Owner name: AMEDITECH INC., CALIFORNIA

Free format text: NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT;REEL/FRAME:036011/0581

Effective date: 20150618

Owner name: ALERE SCARBOROUGH, INC., MAINE

Free format text: NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT;REEL/FRAME:036011/0581

Effective date: 20150618

Owner name: APPLIED BIOTECH, INC., CALIFORNIA

Free format text: NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT;REEL/FRAME:036011/0581

Effective date: 20150618

Owner name: INSTANT TECHNOLOGIES, INC., VIRGINIA

Free format text: NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT;REEL/FRAME:036011/0581

Effective date: 20150618

Owner name: ZYCARE, INC., NORTH CAROLINA

Free format text: NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT;REEL/FRAME:036011/0581

Effective date: 20150618

Owner name: MATRITECH, INC., MASSACHUSETTS

Free format text: NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT;REEL/FRAME:036011/0581

Effective date: 20150618

Owner name: ALERE SAN DIEGO, INC., CALIFORNIA

Free format text: NOTICE OF RELEASE OF SECURITY INTEREST IN PATENTS RECORDED AT REEL 026557 FRAME 0287;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS ADMINISTRATIVE AGENT AND COLLATERAL AGENT;REEL/FRAME:036011/0581

Effective date: 20150618

AS Assignment

Owner name: GENERAL ELECTRIC CAPITAL CORPORATION, AS COLLATERAL AGENT, MARYLAND

Free format text: INTELLECTUAL PROPERTY SECURITY AGREEMENT;ASSIGNORS:ALERE CONNECT, LLC;ALERE SAN DIEGO, INC. (FKA BIOSITE INC. OR FKA CHOLESTECH CORP. OR FKA HEMOSENSE INC. OR FKA INVERNESS MEDICAL-BIOSTAR INC. OR FKA ISCHEMIA TECHNOLOGIES, INC. OR FKA TWISTDX, INC.);ALERE SCARBOROUGH, INC. (FKA MATRITECH, INC. FKA ADVANTAGE DIAGNOSTICS CORP. OR FKA BINAX, INC. OR FKA MILANO ACQUISITION CORP.);AND OTHERS;REEL/FRAME:036994/0192

Effective date: 20150618

Owner name: GENERAL ELECTRIC CAPITAL CORPORATION, AS COLLATERA

Free format text: INTELLECTUAL PROPERTY SECURITY AGREEMENT;ASSIGNORS:ALERE CONNECT, LLC;ALERE SAN DIEGO, INC. (FKA BIOSITE INC. OR FKA CHOLESTECH CORP. OR FKA HEMOSENSE INC. OR FKA INVERNESS MEDICAL-BIOSTAR INC. OR FKA ISCHEMIA TECHNOLOGIES, INC. OR FKA TWISTDX, INC.);ALERE SCARBOROUGH, INC. (FKA MATRITECH, INC. FKA ADVANTAGE DIAGNOSTICS CORP. OR FKA BINAX, INC. OR FKA MILANO ACQUISITION CORP.);AND OTHERS;REEL/FRAME:036994/0192

Effective date: 20150618

AS Assignment

Owner name: HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS SUCCESSOR ADMINISTRATIVE AGENT, MARYLAND

Free format text: ASSIGNMENT OF IP SECURITY AGREEMENT, PREVIOUSLY RECORDED AT REEL 036994, FRAME 0192;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS RETIRING ADMINISTRATIVE AGENT;REEL/FRAME:037115/0498

Effective date: 20151113

Owner name: HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS SUCCESSOR

Free format text: ASSIGNMENT OF IP SECURITY AGREEMENT, PREVIOUSLY RECORDED AT REEL 036994, FRAME 0192;ASSIGNOR:GENERAL ELECTRIC CAPITAL CORPORATION, AS RETIRING ADMINISTRATIVE AGENT;REEL/FRAME:037115/0498

Effective date: 20151113

AS Assignment

Owner name: ALERE SCARBOROUGH, INC. (FKA MATRITECH, INC. FKA ADVANTAGE DIAGNOSTICS CORP. OR FKA BINAX, INC. OR FKA MILANO ACQUISITION CORP.), MAINE

Free format text: RELEASE OF SECURITY INTEREST IN INTELLECTUAL PROPERTY RECORDED AT REEL 036994, FRAME 0192 AND REEL 037115, FRAME 0498;ASSIGNOR:HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS COLLATERAL AGENT;REEL/FRAME:044213/0258

Effective date: 20171003

Owner name: INNOVACON, INC. (FKA APPLIED BIOTECH, INC. OR FKA AMEDITECH INC.), CALIFORNIA

Free format text: RELEASE OF SECURITY INTEREST IN INTELLECTUAL PROPERTY RECORDED AT REEL 036994, FRAME 0192 AND REEL 037115, FRAME 0498;ASSIGNOR:HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS COLLATERAL AGENT;REEL/FRAME:044213/0258

Effective date: 20171003

Owner name: IONIAN TECHNOLOGIES, LLC (FKA IONIAN TECHNOLOGIES, INC.), CALIFORNIA

Free format text: RELEASE OF SECURITY INTEREST IN INTELLECTUAL PROPERTY RECORDED AT REEL 036994, FRAME 0192 AND REEL 037115, FRAME 0498;ASSIGNOR:HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS COLLATERAL AGENT;REEL/FRAME:044213/0258

Effective date: 20171003

Owner name: QUALITY ASSURED SERVICES INC. (FKA ZYCARE INC.), FLORIDA

Free format text: RELEASE OF SECURITY INTEREST IN INTELLECTUAL PROPERTY RECORDED AT REEL 036994, FRAME 0192 AND REEL 037115, FRAME 0498;ASSIGNOR:HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS COLLATERAL AGENT;REEL/FRAME:044213/0258

Effective date: 20171003

Owner name: ALERE SAN DIEGO, INC. (FKA BIOSITE INC. OR FKA CHOLESTECH CORP. OR FKA HEMOSENSE INC. OR FKA INVERNESS MEDICAL-BIOSTAR INC. OR FKA ISCHEMIA TECHNOLOGIES, INC. OR FKA TWISTDX, INC.), CALIFORNIA

Free format text: RELEASE OF SECURITY INTEREST IN INTELLECTUAL PROPERTY RECORDED AT REEL 036994, FRAME 0192 AND REEL 037115, FRAME 0498;ASSIGNOR:HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS COLLATERAL AGENT;REEL/FRAME:044213/0258

Effective date: 20171003

Owner name: IONIAN TECHNOLOGIES, LLC (FKA IONIAN TECHNOLOGIES,

Free format text: RELEASE OF SECURITY INTEREST IN INTELLECTUAL PROPERTY RECORDED AT REEL 036994, FRAME 0192 AND REEL 037115, FRAME 0498;ASSIGNOR:HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS COLLATERAL AGENT;REEL/FRAME:044213/0258

Effective date: 20171003

Owner name: ALERE SCARBOROUGH, INC. (FKA MATRITECH, INC. FKA A

Free format text: RELEASE OF SECURITY INTEREST IN INTELLECTUAL PROPERTY RECORDED AT REEL 036994, FRAME 0192 AND REEL 037115, FRAME 0498;ASSIGNOR:HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS COLLATERAL AGENT;REEL/FRAME:044213/0258

Effective date: 20171003

Owner name: INNOVACON, INC. (FKA APPLIED BIOTECH, INC. OR FKA

Free format text: RELEASE OF SECURITY INTEREST IN INTELLECTUAL PROPERTY RECORDED AT REEL 036994, FRAME 0192 AND REEL 037115, FRAME 0498;ASSIGNOR:HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS COLLATERAL AGENT;REEL/FRAME:044213/0258

Effective date: 20171003

Owner name: STANDING STONE, LLC, CONNECTICUT

Free format text: RELEASE OF SECURITY INTEREST IN INTELLECTUAL PROPERTY RECORDED AT REEL 036994, FRAME 0192 AND REEL 037115, FRAME 0498;ASSIGNOR:HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS COLLATERAL AGENT;REEL/FRAME:044213/0258

Effective date: 20171003

Owner name: ALERE SAN DIEGO, INC. (FKA BIOSITE INC. OR FKA CHO

Free format text: RELEASE OF SECURITY INTEREST IN INTELLECTUAL PROPERTY RECORDED AT REEL 036994, FRAME 0192 AND REEL 037115, FRAME 0498;ASSIGNOR:HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS COLLATERAL AGENT;REEL/FRAME:044213/0258

Effective date: 20171003

Owner name: ALERE CONNECT, LLC, ARIZONA

Free format text: RELEASE OF SECURITY INTEREST IN INTELLECTUAL PROPERTY RECORDED AT REEL 036994, FRAME 0192 AND REEL 037115, FRAME 0498;ASSIGNOR:HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS COLLATERAL AGENT;REEL/FRAME:044213/0258

Effective date: 20171003

Owner name: ESCREEN, INC., KANSAS

Free format text: RELEASE OF SECURITY INTEREST IN INTELLECTUAL PROPERTY RECORDED AT REEL 036994, FRAME 0192 AND REEL 037115, FRAME 0498;ASSIGNOR:HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS COLLATERAL AGENT;REEL/FRAME:044213/0258

Effective date: 20171003

Owner name: QUALITY ASSURED SERVICES INC. (FKA ZYCARE INC.), F

Free format text: RELEASE OF SECURITY INTEREST IN INTELLECTUAL PROPERTY RECORDED AT REEL 036994, FRAME 0192 AND REEL 037115, FRAME 0498;ASSIGNOR:HEALTHCARE FINANCIAL SOLUTIONS, LLC, AS COLLATERAL AGENT;REEL/FRAME:044213/0258

Effective date: 20171003

MAFP Maintenance fee payment

Free format text: PAYMENT OF MAINTENANCE FEE, 12TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1553)

Year of fee payment: 12

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载